WO2012096329A1 - Method for detecting single nucleotide polymorphisms - Google Patents

Method for detecting single nucleotide polymorphisms Download PDF

Info

Publication number
WO2012096329A1
WO2012096329A1 PCT/JP2012/050430 JP2012050430W WO2012096329A1 WO 2012096329 A1 WO2012096329 A1 WO 2012096329A1 JP 2012050430 W JP2012050430 W JP 2012050430W WO 2012096329 A1 WO2012096329 A1 WO 2012096329A1
Authority
WO
WIPO (PCT)
Prior art keywords
single nucleotide
group
pcr
guanidine
mutant
Prior art date
Application number
PCT/JP2012/050430
Other languages
French (fr)
Japanese (ja)
Inventor
卓也 與谷
英司 清遠
牛澤　幸司
Original Assignee
積水メディカル株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 積水メディカル株式会社 filed Critical 積水メディカル株式会社
Priority to EP12734188.1A priority Critical patent/EP2664917B1/en
Priority to KR1020137020841A priority patent/KR102047650B1/en
Priority to CN201280005010.3A priority patent/CN103348242B/en
Priority to US13/979,256 priority patent/US20140147842A1/en
Priority to JP2012552752A priority patent/JP6061381B2/en
Publication of WO2012096329A1 publication Critical patent/WO2012096329A1/en
Priority to US14/669,735 priority patent/US9447460B2/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/10—Detection mode being characterised by the assay principle
    • C12Q2565/137—Chromatographic separation

Definitions

  • the present invention relates to a rapid and simple method for detecting a single nucleotide polymorphism.
  • a primer is set at the common sequence site, and polymorphism is present inside the PCR amplification product, that is, within the PCR amplification product.
  • the amplified PCR product is cleaved with a restriction enzyme, and the presence or absence of a polymorphism is determined by the length of the fragment.
  • a restriction enzyme since a restriction enzyme is used, there are problems such as an increase in analysis cost and a long analysis time.
  • the difference in chain length is detected by electrophoresis, there are problems such as complicated operations and a long analysis time.
  • Non-Patent Document 1 discloses a method for separating nucleic acid-related compounds by high performance liquid chromatography. However, even the method disclosed in Non-Patent Document 1 has a problem that it is difficult to sufficiently separate nucleic acids having different chain length differences such as single nucleotide polymorphisms.
  • An object of the present invention is to provide a rapid and simple method for detecting a single nucleotide polymorphism.
  • the present invention is a single nucleotide polymorphism detection method in which wild-type and mutant products amplified by the AS-PCR method are analyzed using ion-exchange chromatography.
  • the present invention is described in detail below.
  • the present inventors have found that single nucleotide polymorphisms can be detected quickly and easily by analyzing the wild-type and mutant products amplified by the AS-PCR method using ion exchange chromatography.
  • the invention has been completed.
  • the AS-PCR (Allele Specific-PCR) method is a method for detecting a gene polymorphism (particularly a single nucleotide polymorphism) using a sequence-specific amplification reaction. Specifically, PCR is performed so that the nucleotide sequence of the single nucleotide polymorphism to be detected is the 3 'end of the primer. When the sequence of the target nucleic acid and the primer are completely complementary, an extension reaction occurs by DNA polymerase. On the other hand, when the sequence of the target nucleic acid and the primer are incompletely complementary, the extension reaction of DNA polymerase is inhibited.
  • this is a method for determining a single nucleotide polymorphism based on the result of an amplification reaction using two kinds of primers having a single nucleotide polymorphism wild-type or mutant nucleotide sequence at the 3 'end.
  • AS-PCR method the method disclosed in “Nature, 324, p.163-166, 1986” can be used.
  • ion exchange chromatography is used. It is preferable that the eluent used for ion exchange chromatography contains a guanidine salt derived from guanidine represented by the following formula (1).
  • guanidine salts include guanidine hydrochloride, guanidine sulfate, guanidine nitrate, guanidine carbonate, guanidine phosphate, guanidine thiocyanate, guanidine sulfamate, aminoguanidine hydrochloride, aminoguanidine bicarbonate, and the like. It is done. Of these, guanidine hydrochloride and guanidine sulfate are preferably used.
  • the concentration of the guanidine salt in the eluent at the time of analysis may be appropriately adjusted according to the substance to be detected, but is desirably 2000 mmol / L or less. Specifically, a method of performing gradient elution with a guanidine salt concentration in the range of 0 to 2000 mmol / L can be mentioned. Therefore, the concentration of guanidine salt at the start of analysis need not be 0 mmol / L, and the salt concentration of guanidine salt at the end of analysis need not be 2000 mmol / L.
  • the gradient elution method may be a low pressure gradient method or a high pressure gradient method, but a method of eluting while performing precise concentration adjustment by the high pressure gradient method is preferred.
  • the guanidine salt may be added to the eluent alone or in combination with other salts.
  • the salt that can be used in combination with the guanidine salt include, for example, a salt composed of a halide such as sodium chloride, potassium chloride, sodium bromide, potassium bromide and an alkali metal, calcium chloride, calcium bromide, magnesium chloride.
  • salts of halides such as magnesium bromide and alkaline earth metals, and inorganic acid salts such as sodium perchlorate, potassium perchlorate, sodium sulfate, potassium sulfate, ammonium sulfate, sodium nitrate, potassium nitrate, etc.
  • organic acid salts such as sodium acetate, potassium acetate, sodium succinate, potassium succinate and the like may be used.
  • buffers and organic solvents can be used. Specifically, for example, Tris hydrochloric acid buffer, TE buffer composed of Tris and EDTA, Tris, acetic acid and EDTA are used. And a TBA buffer solution consisting of Tris, boric acid and EDTA.
  • the pH of the eluent is not particularly limited as long as the nucleic acid chain can be separated by anion exchange.
  • those having a cationic group introduced on at least the surface of the base particle are preferable, and those having a strong cationic group and a weak anionic group on at least the surface of the base particle. Is more preferable.
  • the “strong cationic group” means a cationic group that dissociates in a wide range of pH from 1 to 14. That is, the strong cationic group can be kept dissociated (cationized) without being affected by the pH of the aqueous solution.
  • a quaternary ammonium group is mentioned as a strong cationic group.
  • Specific examples include trialkylammonium groups such as a trimethylammonium group, a triethylammonium group, and a dimethylethylammonium group.
  • examples of counter ions of strong cationic groups include halide ions such as chloride ions, bromide ions, and iodide ions.
  • the amount of the strong cationic group is not particularly limited, but the preferable lower limit per dry weight of the filler is 1 ⁇ eq / g, and the preferable upper limit is 500 ⁇ eq / g.
  • the amount of the strong cationic group is less than 1 ⁇ eq / g, the holding power of the filler is weakened, and the separation performance may be deteriorated.
  • the amount of the strong cationic group exceeds 500 ⁇ eq / g, the retention of the filler becomes too strong, the detection target substance cannot be easily eluted, and problems such as a long analysis time may occur.
  • the “weak anionic group” means an anionic group having a pKa of 3 or more. That is, the weak anionic group is affected by the pH of the aqueous solution, and the dissociation state changes. When the pH is higher than 3, the protons of the carboxy group are dissociated and the proportion of negative charges increases. On the other hand, when the pH is lower than 3, the proportion of the non-dissociated state in which the carboxy group protons are bonded increases.
  • the weak anionic group include a carboxy group and a phosphate group. Of these, a carboxy group is preferable.
  • Examples of a method for introducing a carboxy group into at least the surface of the base particle include, for example, a method of copolymerizing a monomer having a carboxy group, a method of hydrolyzing an ester portion in the monomer, and a carboxy group by treatment with ozone water.
  • a method of forming a carboxy group by ozone gas, a method of forming a carboxy group by plasma treatment, a method of reacting a silane coupling agent having a carboxy group, an epoxy group by copolymerizing a monomer having an epoxy group A known method such as a method of forming a carboxy group by ring opening of can be used.
  • the base particle has a hydrophobic structure portion, particularly a carbon-carbon double bond, it is preferable to use a method of forming a carboxy group by treatment with ozone water.
  • Ozone is highly reactive with a double bond, and ozone that has reacted with the double bond forms an ozonide that is an intermediate, and then a carboxy group or the like is formed.
  • Ozone water means that ozone gas is dissolved in water.
  • the particle surface can be easily oxidized simply by dispersing the particles in ozone water.
  • hydrophilic groups such as a carboxy group, a hydroxyl group, an aldehyde group, and a keto group are formed.
  • Ozone has a strong oxidizing effect, but by treating with ozone water, the particle surface can be oxidized more uniformly than by treating with ozone gas, and carboxy groups are formed more uniformly. preferable.
  • concentration of the dissolved ozone in ozone water is not specifically limited, A preferable minimum is 20 ppm.
  • concentration of dissolved ozone is less than 20 ppm, it takes a long time to form a carboxy group, or the formation of a carboxy group becomes insufficient, and the nonspecific adsorption of the detection target substance is sufficiently suppressed. I cannot do it.
  • a more preferable lower limit of the concentration of dissolved ozone is 50 ppm.
  • ozone water is a method in which raw water and ozone gas are brought into contact with each other through an ozone gas permeable film that allows only gas to pass and blocks liquid from passing through. Can be prepared.
  • the carboxy group introduced on the surface of the substrate particle is almost dissociated, and it is considered that a weak cation exchange interaction occurs with a few cations in the nucleobase.
  • hydrophilic groups such as hydroxyl groups, aldehyde groups and keto groups are formed in addition to carboxy groups, and the presence of these hydrophilic groups acts between the surface of the filler and the nucleic acid. It is thought that the hydrophobic interaction is weakened.
  • the amount of the weak anionic group introduced into at least the surface of the base particle is not particularly limited as long as it is equal to or less than the strong cationic group amount.
  • the base particles for example, synthetic polymer fine particles obtained using a polymerizable monomer, inorganic fine particles such as silica-based particles can be used, and hydrophobic crosslinked polymer particles made of organic synthetic polymers. And a layer made of a hydrophilic polymer having an ion exchange group copolymerized on the surface of the hydrophobic crosslinked polymer particles.
  • Hydrophobic crosslinked polymer is a hydrophobic crosslinked polymer obtained by homopolymerizing one kind of hydrophobic crosslinkable monomer, and obtained by copolymerizing two or more kinds of hydrophobic crosslinkable monomers. Any of a crosslinked polymer and a hydrophobic crosslinked polymer obtained by copolymerizing at least one hydrophobic crosslinking monomer and at least one hydrophobic non-crosslinking monomer may be used.
  • the hydrophobic crosslinkable monomer is not particularly limited as long as it has two or more vinyl groups in one monomer molecule.
  • ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate , Propylene glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate and other di (meth) acrylates tetramethylol methane tri (meth) acrylate, trimethylol propane tri (meth) acrylate, tetramethylol methane tetra
  • Examples include tri (meth) acrylic acid esters such as (meth) acrylate or tetra (meth) acrylic acid esters, and aromatic compounds such as divinylbenzene, divinyltoluene, divinylxylene, and divinylnaphthalene.
  • (meth) acryl means “acryl or methacryl”
  • (meth) acrylate” means “acrylate or
  • the hydrophobic non-crosslinkable monomer is not particularly limited as long as it is a non-crosslinkable polymerizable organic monomer having hydrophobic properties.
  • methyl (meth) acrylate, ethyl (meth) acrylate, propyl examples thereof include (meth) acrylates such as (meth) acrylate, isopropyl (meth) acrylate, butyl (meth) acrylate and t-butyl (meth) acrylate, and styrene monomers such as styrene and methylstyrene.
  • the hydrophobic cross-linked polymer consists of a copolymer of a hydrophobic cross-linkable monomer and a hydrophobic non-cross-linkable monomer
  • the inclusion of segments derived from the hydrophobic cross-linkable monomer in the hydrophobic cross-linked polymer is 10% by weight, and the more preferable lower limit is 20% by weight.
  • the hydrophilic polymer having an ion exchange group is composed of a hydrophilic monomer having an ion exchange group, and includes a segment derived from a hydrophilic monomer having one or more ion exchange groups.
  • Good That is, as a method for producing a hydrophilic polymer having an ion exchange group, a method in which a hydrophilic monomer having an ion exchange group is polymerized alone, a hydrophilic monomer having an ion exchange group and an ion exchange group are used. And a method of copolymerizing with a hydrophilic monomer not to be used.
  • the hydrophilic monomer having an ion exchange group preferably has a strong cationic group, and more preferably has a quaternary ammonium group.
  • Examples include acrylamidoethyltrimethylammonium chloride, acrylamidoethyltriethylammonium chloride, and acrylamidoethyldimethylethylammonium chloride.
  • the average particle diameter of the filler is not particularly limited, but a preferable lower limit is 0.1 ⁇ m and a preferable upper limit is 20 ⁇ m.
  • a preferable lower limit is 0.1 ⁇ m and a preferable upper limit is 20 ⁇ m.
  • the average particle size of the packing material is less than 0.1 ⁇ m, the internal pressure of the column becomes high, which may cause poor separation. If the average particle size of the packing material exceeds 20 ⁇ m, the dead volume in the column becomes too large and may cause poor separation.
  • an average particle diameter shows a volume average particle diameter, and can be measured using a particle size distribution measuring apparatus (made by AccuSizer780 / Particle Sizing Systems).
  • the size of the product amplified by the AS-PCR method is preferably 200 bp or less. If the size of the product amplified by the AS-PCR method exceeds 200 bp, the PCR amplification time or the analysis time in ion exchange chromatography may become long, or sufficient separation performance may not be obtained.
  • the size of the product amplified by the AS-PCR method is more preferably 100 bp or less.
  • the difference in product size (chain length difference) between the wild type and the mutant type amplified by the AS-PCR method is preferably 10 bp or less. Even if the AS primer is designed so that the difference in size between the amplified wild-type product and the mutant product exceeds 10 bp, a desired amplification product may not be obtained in a non-specific amplification reaction or the like.
  • a rapid and simple method for detecting a single nucleotide polymorphism can be provided.
  • Example 1 it is the chromatogram obtained by isolate
  • FIG. 1 it is the chromatogram obtained by isolate
  • FIG. 1 it is a chromatogram obtained by separating and detecting wild type 271 bp and mutant type 274 bp in the UGT1A1 * 6 region using an anion exchange column 1.
  • Reference Example 1 it is a chromatogram obtained by separating and detecting wild type 271 bp and mutant type 274 bp in the UGT1A1 * 6 region using an anion exchange column 2.
  • Reference Example 2 it is a chromatogram obtained by separating and detecting wild type 76 bp and mutant type 79 bp in the UGT1A1 * 6 region using the anion exchange column 1.
  • ozone water is 400 hollow-tube ozone gas permeable membranes having an inner diameter of 0.5 mm, a thickness of 0.04 mm, and a length of 350 cm made of perfluoroalkoxy resin in a jacket having a cylindrical shape with an inner diameter of 15 cm and a length of 20 cm. It was prepared using an ozone water production system (manufactured by Sekisui Chemical Co., Ltd.) containing the accommodated ozone dissolution module. The obtained filler for ion exchange chromatography was measured using a particle size distribution analyzer (Particulate Sizing Systems, “Accurizer 780”), and the average particle size was 10 ⁇ m. The following column (anion exchange column 1) was prepared using the obtained packing material for ion exchange chromatography. Column size: inner diameter 4.6 mm x 20 mm Ion exchange group: quaternary ammonium group
  • Example 1 In Example 1, separation detection of wild type 76 bp and mutant type 79 bp of the UGT1A1 * 6 region was performed.
  • reaction C1000 Bio-Rad Laboratories
  • the temperature cycle is as follows.
  • the template was thermally denatured at 94 ° C. for 30 seconds, 40 cycles of 94 ° C. for 15 seconds, 62 ° C. for 15 seconds, and 68 ° C. for 30 seconds were performed, and finally the temperature was kept at 68 ° C. for 5 minutes. Samples were stored at 4 ° C. until use.
  • Reference Example 1 separation detection of wild type 271 bp and mutant type 274 bp in the UGT1A1 * 6 region was performed.
  • reaction C1000 Bio-Rad Laboratories
  • the temperature cycle is as follows.
  • the template was thermally denatured at 94 ° C. for 30 seconds, 40 cycles of 94 ° C. for 15 seconds, 62 ° C. for 15 seconds, and 68 ° C. for 30 seconds were performed, and finally the temperature was kept at 68 ° C. for 5 minutes. Samples were stored at 4 ° C. until use.
  • Comparative Example 1 In Comparative Example 1, an attempt was made to separate and detect wild type 76 bp and mutant type 96 bp of the UGT1A1 * 6 region.
  • reaction C1000 Bio-Rad Laboratories
  • the temperature cycle is as follows.
  • the template was thermally denatured at 94 ° C. for 30 seconds, 40 cycles of 94 ° C. for 15 seconds, 62 ° C. for 15 seconds, and 68 ° C. for 30 seconds were performed, and finally the temperature was kept at 68 ° C. for 5 minutes. Samples were stored at 4 ° C. until use.
  • Reference Example 2 separation detection of wild type 76 bp and mutant type 79 bp in the UGT1A1 * 6 region was performed.
  • HPLC analysis was performed using anion exchange column 2 in the same manner as in Example 1 except that sodium chloride was used instead of guanidine hydrochloride as the salt added to eluent B.
  • Example 1 chromatograms obtained by separating and detecting wild type 76 bp and mutant type 79 bp of UGT1A1 * 6 region are shown in FIG. 1 (when anion exchange column 1 is used) and FIG. When used). From the results shown in FIGS. 1 and 2, both columns were able to successfully separate and detect wild-type 76 bp and mutant-type 79 bp of UGT1A1 * 6 region amplified by AS-PCR. In particular, when the anion exchange column 1 was used, almost complete separation and detection could be achieved in a short time.
  • FIG. 3 chromatograms obtained by separating and detecting wild type 271 bp and mutant type 274 bp in the UGT1A1 * 6 region are shown in FIG. 3 (when anion exchange column 1 is used) and FIG. When used). From the results of FIGS. 3 and 4, in contrast to Example 1, it was not possible to separate the wild type 271 bp and the mutant type 274 bp of the UGT1A1 * 6 region amplified by AS-PCR. This is considered to be because the difference in chain length between the wild type and the mutant type was small with respect to the size of the AS-PCR amplification product.
  • FIG. 5 shows a chromatogram obtained by separating and detecting wild type 76 bp and mutant type 79 bp in the UGT1A1 * 6 region using the anion exchange column 2 in Reference Example 2.
  • sodium chloride was added to eluent B instead of guanidine hydrochloride, wild type 76 bp and mutant type 79 bp could not be separated.
  • a rapid and simple method for detecting a single nucleotide polymorphism can be provided.

Abstract

The purpose of the present invention is to provide a method for rapidly and simply detecting single nucleotide polymorphisms. The present invention is a method for detecting single nucleotide polymorphisms in which wild-type and variant products, which have been amplified by AS-PCR, are analyzed using ion-exchange chromatography.

Description

一塩基多型の検出方法Single nucleotide polymorphism detection method
本発明は、迅速かつ簡便な一塩基多型の検出方法に関する。 The present invention relates to a rapid and simple method for detecting a single nucleotide polymorphism.
近年、様々な病気や薬の副作用との関連が明らかとなってきた一塩基多型（ＳＮＰ；Ｓｉｎｇｌｅ　Ｎｕｃｌｅｏｔｉｄｅ　Ｐｏｌｙｍｏｒｐｈｉｓｍ）を解析する技術が開発されており、これらの開発においては、一塩基多型を簡便かつ短時間に精度良く検出することが重要な要素となっている。
一塩基多型を解析する方法として、ＲＦＬＰ法（Ｒｅｓｔｒｉｃｔｉｏｎ　Ｆｒａｇｍｅｔ　Ｌｅｎｇｔｈ　Ｐｏｌｙｍｏｒｐｈｉｓｍ）が知られている。ＲＦＬＰ法は、ＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ　Ｃｈａｉｎ　Ｒｅａｃｔｉｏｎ）増幅産物中の遺伝子変異部位を認識する制限酵素が存在する場合、共通配列部位にプライマーを設定し、その内側、すなわち、ＰＣＲ増幅産物内に多型性をもたせて増幅し、得られたＰＣＲ産物を制限酵素で切断し、その断片の長さにより、多型の有無を判定する方法である。しかしながら、制限酵素を用いるため、分析コストが上がったり、解析全体の時間が長くなったりする等の課題がある。また、電気泳動により鎖長差を検出するため、作業が煩雑になったり、解析全体の時間が長くなったりする等の課題もある。 In recent years, techniques for analyzing single nucleotide polymorphisms (SNPs) that have been associated with various diseases and side effects of drugs have been developed. In these developments, single nucleotide polymorphisms have been developed. It is an important factor that the detection is simple and accurate in a short time.
As a method for analyzing a single nucleotide polymorphism, an RFLP method (Restriction Fragment Length Polymorphism) is known. In the RFLP method, when there is a restriction enzyme that recognizes a gene mutation site in a PCR (Polymerase Chain Reaction) amplification product, a primer is set at the common sequence site, and polymorphism is present inside the PCR amplification product, that is, within the PCR amplification product. In this method, the amplified PCR product is cleaved with a restriction enzyme, and the presence or absence of a polymorphism is determined by the length of the fragment. However, since a restriction enzyme is used, there are problems such as an increase in analysis cost and a long analysis time. In addition, since the difference in chain length is detected by electrophoresis, there are problems such as complicated operations and a long analysis time.
一方、生化学分野や医学分野等において、核酸、タンパク質、多糖類といった生体高分子の分離には、簡便かつ短時間に精度良く検出できる方法としてイオン交換クロマトグラフィーが利用されている。イオン交換クロマトグラフィーを用いると、電気泳動による測定に必要とされるような煩雑な作業が緩和される。非特許文献１には、核酸関連化合物を高速液体クロマトグラフィーで分離する方法が開示されている。しかしながら、非特許文献１に開示された方法でも、一塩基多型のような接近する鎖長差を有する核酸を充分に分離することが難しいという問題がある。 On the other hand, in the biochemical field, the medical field, and the like, ion exchange chromatography is used as a simple and accurate method for detecting biopolymers such as nucleic acids, proteins, and polysaccharides in a short time. When ion exchange chromatography is used, troublesome work required for measurement by electrophoresis is eased. Non-Patent Document 1 discloses a method for separating nucleic acid-related compounds by high performance liquid chromatography. However, even the method disclosed in Non-Patent Document 1 has a problem that it is difficult to sufficiently separate nucleic acids having different chain length differences such as single nucleotide polymorphisms.
本発明は、迅速かつ簡便な一塩基多型の検出方法を提供することを目的とする。 An object of the present invention is to provide a rapid and simple method for detecting a single nucleotide polymorphism.
本発明は、ＡＳ－ＰＣＲ法によって増幅された野生型と変異型の産物を、イオン交換クロマトグラフィーを用いて分析する一塩基多型の検出方法である。
以下に本発明を詳述する。 The present invention is a single nucleotide polymorphism detection method in which wild-type and mutant products amplified by the AS-PCR method are analyzed using ion-exchange chromatography.
The present invention is described in detail below.
本発明者らは、ＡＳ－ＰＣＲ法によって増幅された野生型と変異型の産物を、イオン交換クロマトグラフィーを用いて分析することによって、迅速かつ簡便に一塩基多型を検出できることを見出し、本発明を完成させるに至った。 The present inventors have found that single nucleotide polymorphisms can be detected quickly and easily by analyzing the wild-type and mutant products amplified by the AS-PCR method using ion exchange chromatography. The invention has been completed.
ＡＳ－ＰＣＲ（Ａｌｌｅｌｅ　Ｓｐｅｃｉｆｉｃ－ＰＣＲ）法は、配列特異的な増幅反応を利用した遺伝子多型（特に一塩基多型）を検出する方法である。具体的には、検出したい一塩基多型の塩基配列がプライマーの３’末端になるようにしてＰＣＲを行う。標的核酸の配列とプライマーが完全に相補である場合、ＤＮＡポリメラーゼにより伸長反応が起こる。一方、標的核酸の配列とプライマーとが不完全相補である場合には、ＤＮＡポリメラーゼの伸長反応が阻害される。このように、一塩基多型の野生型又は変異型の塩基配列を３’末端に有する２種類のプライマーを使用し、増幅反応の結果に基づいて一塩基多型の判定を行う方法である。ＡＳ－ＰＣＲ法としては、「Ｎａｔｕｒｅ，３２４，ｐ．１６３－１６６，１９８６」に開示されている方法を用いることができる。 The AS-PCR (Allele Specific-PCR) method is a method for detecting a gene polymorphism (particularly a single nucleotide polymorphism) using a sequence-specific amplification reaction. Specifically, PCR is performed so that the nucleotide sequence of the single nucleotide polymorphism to be detected is the 3 'end of the primer. When the sequence of the target nucleic acid and the primer are completely complementary, an extension reaction occurs by DNA polymerase. On the other hand, when the sequence of the target nucleic acid and the primer are incompletely complementary, the extension reaction of DNA polymerase is inhibited. Thus, this is a method for determining a single nucleotide polymorphism based on the result of an amplification reaction using two kinds of primers having a single nucleotide polymorphism wild-type or mutant nucleotide sequence at the 3 'end. As the AS-PCR method, the method disclosed in “Nature, 324, p.163-166, 1986” can be used.
本発明の一塩基多型の検出方法では、イオン交換クロマトグラフィーを用いる。
イオン交換クロマトグラフィーに用いる溶離液は、下記式（１）で示されるグアニジンから誘導されるグアニジン塩を含有することが好ましい。 In the method for detecting a single nucleotide polymorphism of the present invention, ion exchange chromatography is used.
It is preferable that the eluent used for ion exchange chromatography contains a guanidine salt derived from guanidine represented by the following formula (1).
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
グアニジン塩としては、例えば、グアニジン塩酸塩、グアニジン硫酸塩、グアニジン硝酸塩、グアニジン炭酸塩、グアニジンリン酸塩、グアニジンチオシアン酸塩、グアニジンスルファミン酸塩、アミノグアニジン塩酸塩、アミノグアニジン重炭酸塩等が挙げられる。なかでも、グアニジン塩酸塩、グアニジン硫酸塩が好適に用いられる。 Examples of guanidine salts include guanidine hydrochloride, guanidine sulfate, guanidine nitrate, guanidine carbonate, guanidine phosphate, guanidine thiocyanate, guanidine sulfamate, aminoguanidine hydrochloride, aminoguanidine bicarbonate, and the like. It is done. Of these, guanidine hydrochloride and guanidine sulfate are preferably used.
溶離液におけるグアニジン塩の分析時の濃度は、検出対象物質に合わせて、適宜調整すればよいが、２０００ｍｍｏｌ／Ｌ以下であることが望ましい。
具体的には、グアニジン塩の濃度を０～２０００ｍｍｏｌ／Ｌの範囲でグラジエント溶出させる方法を挙げることができる。従って、分析開始時のグアニジン塩の濃度は０ｍｍｏｌ／Ｌである必要はなく、また、分析終了時のグアニジン塩の塩濃度も２０００ｍｍｏｌ／Ｌである必要はない。
グラジエント溶出の方法は、低圧グラジエント法であっても高圧グラジエント法であってもよいが、高圧グラジエント法による精密な濃度調整を行いながら溶出させる方法が好ましい。 The concentration of the guanidine salt in the eluent at the time of analysis may be appropriately adjusted according to the substance to be detected, but is desirably 2000 mmol / L or less.
Specifically, a method of performing gradient elution with a guanidine salt concentration in the range of 0 to 2000 mmol / L can be mentioned. Therefore, the concentration of guanidine salt at the start of analysis need not be 0 mmol / L, and the salt concentration of guanidine salt at the end of analysis need not be 2000 mmol / L.
The gradient elution method may be a low pressure gradient method or a high pressure gradient method, but a method of eluting while performing precise concentration adjustment by the high pressure gradient method is preferred.
グアニジン塩は、溶離液に単独で添加してもよいし、他の塩と組み合わせて添加してもよい。グアニジン塩に組み合わせて用いることができる塩としては、例えば、塩化ナトリウム、塩化カリウム、臭化ナトリウム、臭化カリウム等のハロゲン化物とアルカリ金属とからなる塩や、塩化カルシウム、臭化カルシウム、塩化マグネシウム、臭化マグネシウム等のハロゲン化物とアルカリ土類金属とからなる塩や、過塩素酸ナトリウム、過塩素酸カリウム、硫酸ナトリウム、硫酸カリウム、硫酸アンモニウム、硝酸ナトリウム、硝酸カリウム等の無機酸塩等が挙げられる。また、酢酸ナトリウム、酢酸カリウム、コハク酸ナトリウム、コハク酸カリウム等の有機酸塩を用いてもよい。 The guanidine salt may be added to the eluent alone or in combination with other salts. Examples of the salt that can be used in combination with the guanidine salt include, for example, a salt composed of a halide such as sodium chloride, potassium chloride, sodium bromide, potassium bromide and an alkali metal, calcium chloride, calcium bromide, magnesium chloride. And salts of halides such as magnesium bromide and alkaline earth metals, and inorganic acid salts such as sodium perchlorate, potassium perchlorate, sodium sulfate, potassium sulfate, ammonium sulfate, sodium nitrate, potassium nitrate, etc. . Further, organic acid salts such as sodium acetate, potassium acetate, sodium succinate, potassium succinate and the like may be used.
溶離液に用いる緩衝液としては、公知の緩衝液類や有機溶媒類を用いることができ、具体的には例えば、トリス塩酸緩衝液、トリスとＥＤＴＡとからなるＴＥ緩衝液、トリスと酢酸とＥＤＴＡとからなるＴＡＥ緩衝液、トリスとホウ酸とＥＤＴＡとからなるＴＢＡ緩衝液等が挙げられる。 As the buffer used for the eluent, known buffers and organic solvents can be used. Specifically, for example, Tris hydrochloric acid buffer, TE buffer composed of Tris and EDTA, Tris, acetic acid and EDTA are used. And a TBA buffer solution consisting of Tris, boric acid and EDTA.
溶離液のｐＨは特に制限されず、アニオン交換によって核酸鎖を分離できる範囲であればよい。 The pH of the eluent is not particularly limited as long as the nucleic acid chain can be separated by anion exchange.
イオン交換クロマトグラフィーに用いる充填剤としては、基材粒子の少なくとも表面にカチオン性基が導入されているものが好ましく、基材粒子の少なくとも表面に強カチオン性基と弱アニオン性基とを有するものがより好ましい。 As a filler used for ion exchange chromatography, those having a cationic group introduced on at least the surface of the base particle are preferable, and those having a strong cationic group and a weak anionic group on at least the surface of the base particle. Is more preferable.
本明細書において、「強カチオン性基」とは、ｐＨが１から１４の広い範囲で解離するカチオン性基を意味する。すなわち、強カチオン性基は、水溶液のｐＨに影響を受けず解離した（カチオン化した）状態を保つことが可能である。 In the present specification, the “strong cationic group” means a cationic group that dissociates in a wide range of pH from 1 to 14. That is, the strong cationic group can be kept dissociated (cationized) without being affected by the pH of the aqueous solution.
強カチオン性基としては、４級アンモニウム基が挙げられる。具体的には例えば、トリメチルアンモニウム基、トリエチルアンモニウム基、ジメチルエチルアンモニウム基等のトリアルキルアンモニウム基等が挙げられる。
また、強カチオン性基のカウンターイオンとしては、例えば、塩化物イオン、臭化物イオン、ヨウ化物イオン等のハロゲン化物イオンが挙げられる。 A quaternary ammonium group is mentioned as a strong cationic group. Specific examples include trialkylammonium groups such as a trimethylammonium group, a triethylammonium group, and a dimethylethylammonium group.
In addition, examples of counter ions of strong cationic groups include halide ions such as chloride ions, bromide ions, and iodide ions.
強カチオン性基量は特に限定されないが、充填剤の乾燥重量あたりの好ましい下限は１μｅｑ／ｇ、好ましい上限は５００μｅｑ／ｇである。強カチオン性基量が１μｅｑ／ｇ未満であると、充填剤の保持力が弱くなり、分離性能が悪くなることがある。強カチオン性基量が５００μｅｑ／ｇを超えると、充填剤の保持力が強くなりすぎ、検出対象物質を容易に溶出させることができず、分析時間が長くなる等の問題が生じることがある。 The amount of the strong cationic group is not particularly limited, but the preferable lower limit per dry weight of the filler is 1 μeq / g, and the preferable upper limit is 500 μeq / g. When the amount of the strong cationic group is less than 1 μeq / g, the holding power of the filler is weakened, and the separation performance may be deteriorated. When the amount of the strong cationic group exceeds 500 μeq / g, the retention of the filler becomes too strong, the detection target substance cannot be easily eluted, and problems such as a long analysis time may occur.
本明細書において「弱アニオン性基」とは、ｐＫａが３以上のアニオン性基を意味する。すなわち、上記弱アニオン性基は、水溶液のｐＨによる影響を受け、解離状態が変化する。ｐＨが３より高くなると、カルボキシ基のプロトンは解離し、マイナスの電荷を持つ割合が増える。逆に、ｐＨが３より低くなると、カルボキシ基のプロトンが結合した非解離状態の割合が増える。
上記弱アニオン性基としては、例えば、カルボキシ基、リン酸基等が挙げられる。なかでも、カルボキシ基であることが好ましい。 In the present specification, the “weak anionic group” means an anionic group having a pKa of 3 or more. That is, the weak anionic group is affected by the pH of the aqueous solution, and the dissociation state changes. When the pH is higher than 3, the protons of the carboxy group are dissociated and the proportion of negative charges increases. On the other hand, when the pH is lower than 3, the proportion of the non-dissociated state in which the carboxy group protons are bonded increases.
Examples of the weak anionic group include a carboxy group and a phosphate group. Of these, a carboxy group is preferable.
カルボキシ基を基材粒子の少なくとも表面に導入する方法としては、例えば、カルボキシ基を有する単量体を共重合する方法、単量体中のエステル部を加水分解する方法、オゾン水処理によってカルボキシ基を形成する方法、オゾンガスによってカルボキシ基を形成する方法、プラズマ処理によってカルボキシ基を形成する方法、カルボキシ基を有するシランカップリング剤を反応させる方法、エポキシ基を有する単量体を共重合させエポキシ基の開環によってカルボキシ基を形成する方法等、公知の方法を用いることができる。なかでも、基材粒子が疎水性の構造部分、特に炭素－炭素の二重結合を有するものである場合、オゾン水処理によってカルボキシ基を形成する方法を用いることが好ましい。 Examples of a method for introducing a carboxy group into at least the surface of the base particle include, for example, a method of copolymerizing a monomer having a carboxy group, a method of hydrolyzing an ester portion in the monomer, and a carboxy group by treatment with ozone water. A method of forming a carboxy group by ozone gas, a method of forming a carboxy group by plasma treatment, a method of reacting a silane coupling agent having a carboxy group, an epoxy group by copolymerizing a monomer having an epoxy group A known method such as a method of forming a carboxy group by ring opening of can be used. Among these, when the base particle has a hydrophobic structure portion, particularly a carbon-carbon double bond, it is preferable to use a method of forming a carboxy group by treatment with ozone water.
オゾン水処理によってカルボキシ基を形成する方法について説明する。
オゾンは二重結合との反応性が高く、二重結合と反応したオゾンは、中間体であるオゾナイドを形成し、その後、カルボキシ基等が形成される。 A method for forming a carboxy group by ozone water treatment will be described.
Ozone is highly reactive with a double bond, and ozone that has reacted with the double bond forms an ozonide that is an intermediate, and then a carboxy group or the like is formed.
オゾン水とは、オゾンガスが水に溶解したものを意味する。
オゾン水を用いることにより、オゾン水中に粒子を分散させるだけで粒子表面を簡便に酸化させることができる。その結果、基材粒子における疎水性の構造部分が酸化され、カルボキシ基、水酸基、アルデヒド基、ケト基等の親水性基が形成されると考えられる。
オゾンには強力な酸化作用があるが、オゾン水を用いて処理することにより、オゾンガスを用いて処理するよりも粒子表面を均一に酸化させることができ、より均一にカルボキシ基が形成されるので好ましい。 Ozone water means that ozone gas is dissolved in water.
By using ozone water, the particle surface can be easily oxidized simply by dispersing the particles in ozone water. As a result, it is considered that the hydrophobic structure portion in the base particle is oxidized, and hydrophilic groups such as a carboxy group, a hydroxyl group, an aldehyde group, and a keto group are formed.
Ozone has a strong oxidizing effect, but by treating with ozone water, the particle surface can be oxidized more uniformly than by treating with ozone gas, and carboxy groups are formed more uniformly. preferable.
オゾン水における溶存オゾンの濃度は特に限定されないが、好ましい下限は２０ｐｐｍである。溶存オゾンの濃度が２０ｐｐｍ未満であると、カルボキシ基を形成するのに長時間を必要としたり、カルボキシ基の形成が不充分となって、検出対象物質の非特異吸着等を充分に抑制することができなかったりする。溶存オゾンの濃度のより好ましい下限は５０ｐｐｍである。 Although the density | concentration of the dissolved ozone in ozone water is not specifically limited, A preferable minimum is 20 ppm. When the concentration of dissolved ozone is less than 20 ppm, it takes a long time to form a carboxy group, or the formation of a carboxy group becomes insufficient, and the nonspecific adsorption of the detection target substance is sufficiently suppressed. I cannot do it. A more preferable lower limit of the concentration of dissolved ozone is 50 ppm.
オゾン水は、例えば、特開２００１－３３０９６９号公報等に記載されているように、原料水とオゾンガスとを、気体のみを透過し液体の透過を阻止するオゾンガス透過膜を介して接触させる方法等により調製することができる。 For example, as described in Japanese Patent Application Laid-Open No. 2001-330969, ozone water is a method in which raw water and ozone gas are brought into contact with each other through an ozone gas permeable film that allows only gas to pass and blocks liquid from passing through. Can be prepared.
アルカリ条件下においては、基材粒子の表面に導入されたカルボキシ基はほぼ解離した状態にあり、核酸塩基中の僅かなカチオンとの間に弱いカチオン交換相互作用が生じると考えられる。
また、オゾン水によって処理することで、カルボキシ基の他、水酸基、アルデヒド基、ケト基等の親水性基が形成され、これらの親水性基の存在によって充填剤の表面と核酸との間に働く疎水性相互作用が弱まると考えられる。
従って、少なくとも表面に強カチオン性基と弱アニオン性基とを有する充填剤を用いた場合、主たる相互作用である充填剤表面と核酸との間に働くアニオン交換相互作用に加え、上述したように、弱いカチオン交換相互作用が働いたり、疎水性相互作用が弱まったりすることによって分離性能が向上するものと考えられる。 Under alkaline conditions, the carboxy group introduced on the surface of the substrate particle is almost dissociated, and it is considered that a weak cation exchange interaction occurs with a few cations in the nucleobase.
Moreover, by treating with ozone water, hydrophilic groups such as hydroxyl groups, aldehyde groups and keto groups are formed in addition to carboxy groups, and the presence of these hydrophilic groups acts between the surface of the filler and the nucleic acid. It is thought that the hydrophobic interaction is weakened.
Therefore, when using a filler having at least a strong cationic group and a weak anionic group on the surface, in addition to the anion exchange interaction acting between the filler surface and the nucleic acid, which is the main interaction, as described above It is considered that the separation performance is improved by the weak cation exchange interaction or the hydrophobic interaction being weakened.
基材粒子の少なくとも表面に導入される弱アニオン性基量は、強カチオン性基量以下であれば特に限定されない。 The amount of the weak anionic group introduced into at least the surface of the base particle is not particularly limited as long as it is equal to or less than the strong cationic group amount.
基材粒子としては、例えば、重合性単量体等を用いて得られる合成高分子微粒子、シリカ系等の無機微粒子等を用いることができるが、有機合成高分子からなる疎水性架橋重合体粒子と、該疎水性架橋重合体粒子の表面に共重合されたイオン交換基を有する親水性重合体からなる層とからなるものであることが好適である。 As the base particles, for example, synthetic polymer fine particles obtained using a polymerizable monomer, inorganic fine particles such as silica-based particles can be used, and hydrophobic crosslinked polymer particles made of organic synthetic polymers. And a layer made of a hydrophilic polymer having an ion exchange group copolymerized on the surface of the hydrophobic crosslinked polymer particles.
疎水性架橋重合体は、１種の疎水性架橋性単量体を単独重合して得られる疎水性架橋重合体、２種以上の疎水性架橋性単量体を共重合して得られる疎水性架橋重合体、少なくとも１種の疎水性架橋性単量体と少なくとも１種の疎水性非架橋性単量体とを共重合して得られる疎水性架橋重合体のいずれであってもよい。 Hydrophobic crosslinked polymer is a hydrophobic crosslinked polymer obtained by homopolymerizing one kind of hydrophobic crosslinkable monomer, and obtained by copolymerizing two or more kinds of hydrophobic crosslinkable monomers. Any of a crosslinked polymer and a hydrophobic crosslinked polymer obtained by copolymerizing at least one hydrophobic crosslinking monomer and at least one hydrophobic non-crosslinking monomer may be used.
疎水性架橋性単量体としては、単量体１分子中にビニル基を２個以上有するものであれば特に限定されず、例えば、エチレングリコールジ（メタ）アクリレート、ポリエチレングリコールジ（メタ）アクリレート、プロピレングリコールジ（メタ）アクリレート、ポリプロピレングリコールジ（メタ）アクリレート等のジ（メタ）アクリル酸エステルや、テトラメチロールメタントリ（メタ）アクリレート、トリメチロールプロパントリ（メタ）アクリレート、テトラメチロールメタンテトラ（メタ）アクリレート等のトリ（メタ）アクリル酸エステル又はテトラ（メタ）アクリル酸エステルや、ジビニルベンゼン、ジビニルトルエン、ジビニルキシレン、ジビニルナフタレン等の芳香族系化合物等が挙げられる。
なお、本明細書において、「（メタ）アクリル」とは、「アクリル又はメタクリル」を意味し、「（メタ）アクリレート」とは、「アクリレート又はメタクリレート」を意味する。 The hydrophobic crosslinkable monomer is not particularly limited as long as it has two or more vinyl groups in one monomer molecule. For example, ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate , Propylene glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate and other di (meth) acrylates, tetramethylol methane tri (meth) acrylate, trimethylol propane tri (meth) acrylate, tetramethylol methane tetra ( Examples include tri (meth) acrylic acid esters such as (meth) acrylate or tetra (meth) acrylic acid esters, and aromatic compounds such as divinylbenzene, divinyltoluene, divinylxylene, and divinylnaphthalene.
In the present specification, “(meth) acryl” means “acryl or methacryl”, and “(meth) acrylate” means “acrylate or methacrylate”.
疎水性非架橋性単量体としては、疎水性の性質を有する非架橋性の重合性有機単量体であれば特に限定されず、例えば、メチル（メタ）アクリレート、エチル（メタ）アクリレート、プロピル（メタ）アクリレート、イソプロピル（メタ）アクリレート、ブチル（メタ）アクリレート、ｔ－ブチル（メタ）アクリレート等の（メタ）アクリル酸エステルや、スチレン、メチルスチレン等のスチレン系単量体等が挙げられる。 The hydrophobic non-crosslinkable monomer is not particularly limited as long as it is a non-crosslinkable polymerizable organic monomer having hydrophobic properties. For example, methyl (meth) acrylate, ethyl (meth) acrylate, propyl Examples thereof include (meth) acrylates such as (meth) acrylate, isopropyl (meth) acrylate, butyl (meth) acrylate and t-butyl (meth) acrylate, and styrene monomers such as styrene and methylstyrene.
疎水性架橋重合体が、疎水性架橋性単量体と疎水性非架橋性単量体との共重合からなる場合、疎水性架橋重合体における疎水性架橋性単量体に由来するセグメントの含有割合の好ましい下限は１０重量％、より好ましい下限は２０重量％である。 When the hydrophobic cross-linked polymer consists of a copolymer of a hydrophobic cross-linkable monomer and a hydrophobic non-cross-linkable monomer, the inclusion of segments derived from the hydrophobic cross-linkable monomer in the hydrophobic cross-linked polymer The preferable lower limit of the ratio is 10% by weight, and the more preferable lower limit is 20% by weight.
イオン交換基を有する親水性重合体は、イオン交換基を有する親水性単量体から構成されるものであり、１種以上のイオン交換基を有する親水性単量体に由来するセグメントを含めばよい。即ち、イオン交換基を有する親水性重合体を製造する方法としては、イオン交換基を有する親水性単量体単独で重合させる方法、イオン交換基を有する親水性単量体とイオン交換基を有さない親水性単量体とを共重合させる方法等が挙げられる。 The hydrophilic polymer having an ion exchange group is composed of a hydrophilic monomer having an ion exchange group, and includes a segment derived from a hydrophilic monomer having one or more ion exchange groups. Good. That is, as a method for producing a hydrophilic polymer having an ion exchange group, a method in which a hydrophilic monomer having an ion exchange group is polymerized alone, a hydrophilic monomer having an ion exchange group and an ion exchange group are used. And a method of copolymerizing with a hydrophilic monomer not to be used.
イオン交換基を有する親水性単量体としては、強カチオン性基を有するものであることが好ましく、４級アンモニウム基を有するものであることがより好ましい。具体的には例えば、メタクリル酸エチルトリメチルアンモニウムクロリド、メタクリル酸エチルトリエチルアンモニウムクロリド、メタクリル酸エチルジメチルエチルアンモニウムクロリド、アクリル酸エチルトリメチルアンモニウムクロリド、アクリル酸エチルトリエチルアンモニウムクロリド、アクリル酸エチルジメチルエチルアンモニウムクロリド、アクリルアミドエチルトリメチルアンモニウムクロリド、アクリルアミドエチルトリエチルアンモニウムクロリド、アクリルアミドエチルジメチルエチルアンモニウムクロリド等が挙げられる。 The hydrophilic monomer having an ion exchange group preferably has a strong cationic group, and more preferably has a quaternary ammonium group. Specifically, for example, ethyl trimethylammonium chloride, ethyl triethylammonium chloride, ethyl dimethylethylammonium chloride, ethyltrimethylammonium acrylate, ethyltriethylammonium acrylate, ethyl dimethylethylammonium acrylate, Examples include acrylamidoethyltrimethylammonium chloride, acrylamidoethyltriethylammonium chloride, and acrylamidoethyldimethylethylammonium chloride.
充填剤の平均粒子径は特に限定されないが、好ましい下限は０．１μｍ、好ましい上限は２０μｍである。充填剤の平均粒子径が０．１μｍ未満であると、カラムの内圧が高くなり、分離不良を起こすことがある。充填剤の平均粒子径が２０μｍを超えると、カラム内のデッドボリュームが大きくなりすぎて分離不良を起こすことがある。
なお、本明細書において平均粒子径は体積平均粒子径を示し、粒度分布測定装置（ＡｃｃｕＳｉｚｅｒ７８０／Ｐａｒｔｉｃｌｅ　Ｓｉｚｉｎｇ　Ｓｙｓｔｅｍｓ社製）を用いて測定することができる。 The average particle diameter of the filler is not particularly limited, but a preferable lower limit is 0.1 μm and a preferable upper limit is 20 μm. When the average particle size of the packing material is less than 0.1 μm, the internal pressure of the column becomes high, which may cause poor separation. If the average particle size of the packing material exceeds 20 μm, the dead volume in the column becomes too large and may cause poor separation.
In addition, in this specification, an average particle diameter shows a volume average particle diameter, and can be measured using a particle size distribution measuring apparatus (made by AccuSizer780 / Particle Sizing Systems).
本発明の一塩基多型の検出方法において、ＡＳ－ＰＣＲ法によって増幅される産物の大きさは２００ｂｐ以下であることが好ましい。ＡＳ－ＰＣＲ法によって増幅される産物の大きさが２００ｂｐを超えると、ＰＣＲの増幅時間やイオン交換クロマトグラフィーにおける分析時間が長くなったり、充分な分離性能が得られなかったりすることがある。ＡＳ－ＰＣＲ法によって増幅される産物の大きさは、１００ｂｐ以下であることがより好ましい。 In the method for detecting a single nucleotide polymorphism of the present invention, the size of the product amplified by the AS-PCR method is preferably 200 bp or less. If the size of the product amplified by the AS-PCR method exceeds 200 bp, the PCR amplification time or the analysis time in ion exchange chromatography may become long, or sufficient separation performance may not be obtained. The size of the product amplified by the AS-PCR method is more preferably 100 bp or less.
本発明の一塩基多型の検出方法において、ＡＳ－ＰＣＲ法によって増幅される野生型と変異型との産物の大きさの差（鎖長差）は１０ｂｐ以下であることが好ましい。増幅される野生型と変異型との産物の大きさの差が１０ｂｐを超えるようにＡＳプライマーを設計しても、非特異増幅反応等で所望の増幅産物が得られないことがある。 In the method for detecting a single nucleotide polymorphism of the present invention, the difference in product size (chain length difference) between the wild type and the mutant type amplified by the AS-PCR method is preferably 10 bp or less. Even if the AS primer is designed so that the difference in size between the amplified wild-type product and the mutant product exceeds 10 bp, a desired amplification product may not be obtained in a non-specific amplification reaction or the like.
本発明によれば、迅速かつ簡便な一塩基多型の検出方法を提供することができる。 According to the present invention, a rapid and simple method for detecting a single nucleotide polymorphism can be provided.
実施例１において、アニオン交換カラム１を用いてＵＧＴ１Ａ１＊６領域の野生型７６ｂｐと変異型７９ｂｐとを分離検出して得られたクロマトグラムである。In Example 1, it is the chromatogram obtained by isolate | separating and detecting wild type 76 bp and mutant | variant type 79 bp of UGT1A1 * 6 area | region using the anion exchange column 1. FIG. 実施例１において、アニオン交換カラム２を用いてＵＧＴ１Ａ１＊６領域の野生型７６ｂｐと変異型７９ｂｐとを分離検出して得られたクロマトグラムである。In Example 1, it is the chromatogram obtained by isolate | separating and detecting wild type 76bp and mutant | variant type 79 bp of UGT1A1 * 6 area | region using the anion exchange column 2. FIG. 参考例１において、アニオン交換カラム１を用いてＵＧＴ１Ａ１＊６領域の野生型２７１ｂｐと変異型２７４ｂｐとを分離検出して得られたクロマトグラムである。In Reference Example 1, it is a chromatogram obtained by separating and detecting wild type 271 bp and mutant type 274 bp in the UGT1A1 * 6 region using an anion exchange column 1. 参考例１において、アニオン交換カラム２を用いてＵＧＴ１Ａ１＊６領域の野生型２７１ｂｐと変異型２７４ｂｐとを分離検出して得られたクロマトグラムである。In Reference Example 1, it is a chromatogram obtained by separating and detecting wild type 271 bp and mutant type 274 bp in the UGT1A1 * 6 region using an anion exchange column 2. 参考例２において、アニオン交換カラム１を用いてＵＧＴ１Ａ１＊６領域の野生型７６ｂｐと変異型７９ｂｐとを分離検出して得られたクロマトグラムである。In Reference Example 2, it is a chromatogram obtained by separating and detecting wild type 76 bp and mutant type 79 bp in the UGT1A1 * 6 region using the anion exchange column 1.
以下に実施例を掲げて本発明を更に詳しく説明するが、本発明はこれら実施例のみに限定されない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
（アニオン交換カラムの準備）
（アニオン交換カラム１）
攪拌機付き反応器中にて、３重量％ポリビニルアルコール（日本合成化学社製）水溶液２０００ｍＬに、テトラエチレングリコールジメタクリレート（新中村化学工業社製）３００ｇ、トリエチレングリコールジメタクリレート（新中村化学工業社製）１００ｇ、及び、過酸化ベンゾイル（キシダ化学社製）１．０ｇの混合物を添加した。攪拌しながら加熱し、窒素雰囲気下にて８０℃で１時間重合した。次に、強カチオン性のイオン交換基（４級アンモニウム基）を有する単量体として、メタクリル酸エチルトリメチルアンモニウムクロリド（和光純薬工業社製）１００ｇをイオン交換水に溶解し、得られた溶液を上記反応器中に更に添加した。次いで、攪拌しながら窒素雰囲気下にて８０℃で２時間重合し、重合体組成物を得た。得られた重合体組成物を水及びアセトンで洗浄することにより、基材粒子の表面に４級アンモニウム基を有する親水性の被覆重合体粒子を得た。
得られた被覆重合体粒子１０ｇを溶存オゾン濃度１００ｐｐｍのオゾン水３００ｍＬに浸漬し、３０分間攪拌した。攪拌終了後、遠心分離機（日立製作所社製、「Ｈｉｍａｃ　ＣＲ２０Ｇ」）を用いて遠心分離し、上澄みを除去した。この操作を２回繰り返し、被覆重合体粒子にオゾン水処理を施し、４級アンモニウム基とカルボキシ基が共存するイオン交換クロマトグラフィー用充填剤を得た。
なお、オゾン水は、内径１５ｃｍ×長さ２０ｃｍの円柱形を有する外套内に、パーフルオロアルコキシ樹脂からなる内径０．５ｍｍ×厚さ０．０４ｍｍ×長さ３５０ｃｍの中空管状のオゾンガス透過膜４００本収容されたオゾン溶解モジュールを含むオゾン水製造システム（積水化学工業社製）を用いて調製した。
得られたイオン交換クロマトグラフィー用充填剤について、粒度分布計（Ｐａｒｔｉｃｌｅ　Ｓｉｚｉｎｇ　Ｓｙｓｔｅｍｓ社製、「Ａｃｃｕｓｉｚｅｒ７８０」）を用いて測定したところ、平均粒子径は１０μｍであった。
得られたイオン交換クロマトグラフィー用充填剤を用いて以下のカラム（アニオン交換カラム１）を準備した。
カラムサイズ：内径４．６ｍｍ×２０ｍｍ
イオン交換基：４級アンモニウム基 (Preparation of anion exchange column)
(Anion exchange column 1)
In a reactor equipped with a stirrer, 2000 mL of a 3% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd.), 300 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), triethylene glycol dimethacrylate (Shin Nakamura Chemical Co., Ltd.) 100 g) and 1.0 g of benzoyl peroxide (Kishida Chemical Co., Ltd.) 1.0 g was added. The mixture was heated with stirring and polymerized at 80 ° C. for 1 hour in a nitrogen atmosphere. Next, 100 g of ethyltrimethylammonium methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) as a monomer having a strong cationic ion exchange group (quaternary ammonium group) was dissolved in ion exchange water, and the resulting solution was obtained. Was further added to the reactor. Subsequently, it superposed | polymerized at 80 degreeC under nitrogen atmosphere for 2 hours, stirring, and obtained the polymer composition. The obtained polymer composition was washed with water and acetone to obtain hydrophilic coated polymer particles having a quaternary ammonium group on the surface of the substrate particles.
10 g of the obtained coated polymer particles were immersed in 300 mL of ozone water having a dissolved ozone concentration of 100 ppm and stirred for 30 minutes. After completion of stirring, the mixture was centrifuged using a centrifuge (“Himac CR20G” manufactured by Hitachi, Ltd.), and the supernatant was removed. This operation was repeated twice, and the coated polymer particles were treated with ozone water to obtain a packing material for ion exchange chromatography in which a quaternary ammonium group and a carboxy group coexist.
In addition, ozone water is 400 hollow-tube ozone gas permeable membranes having an inner diameter of 0.5 mm, a thickness of 0.04 mm, and a length of 350 cm made of perfluoroalkoxy resin in a jacket having a cylindrical shape with an inner diameter of 15 cm and a length of 20 cm. It was prepared using an ozone water production system (manufactured by Sekisui Chemical Co., Ltd.) containing the accommodated ozone dissolution module.
The obtained filler for ion exchange chromatography was measured using a particle size distribution analyzer (Particulate Sizing Systems, “Accurizer 780”), and the average particle size was 10 μm.
The following column (anion exchange column 1) was prepared using the obtained packing material for ion exchange chromatography.
Column size: inner diameter 4.6 mm x 20 mm
Ion exchange group: quaternary ammonium group
（アニオン交換カラム２）
市販されているカラムとして、以下のカラムを準備した。
品名：ＴＳＫ－ｇｅｌ　ＤＮＡ－ＳＴＡＴ（東ソー社製）
カラムサイズ：内径４．６ｍｍ×長さ１００ｍｍ
イオン交換基：４級アンモニウム基 (Anion exchange column 2)
The following columns were prepared as commercially available columns.
Product name: TSK-gel DNA-STAT (manufactured by Tosoh Corporation)
Column size: inner diameter 4.6 mm x length 100 mm
Ion exchange group: quaternary ammonium group
（実施例１）
実施例１では、ＵＧＴ１Ａ１＊６領域の野生型７６ｂｐと変異型７９ｂｐとの分離検出を行った。 Example 1
In Example 1, separation detection of wild type 76 bp and mutant type 79 bp of the UGT1A1 * 6 region was performed.
（ＡＳ－ＰＣＲ増幅）
以下に示すＡＳ－ＰＣＲ条件によって野生型と変異型の増幅産物を得た。
（１）試薬
ＡｃｃｕＰｒｉｍｅ　Ｔａｑ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　Ｈｉｇｈ　Ｆｉｄｅｌｉｔｙ（Ｉｎｖｉｔｏｒｇｅｎ社製、Ｌｏｔ．７６０８１６）
１０×ＡｃｃｕＰｒｉｍｅ　ＰＣＲ　Ｂｕｆｆｅｒ　Ｉ
ＡｃｃｕＰｒｉｍｅ　Ｔａｑ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　Ｈｉｇｈ　Ｆｉｄｅｌｉｔｙ（５Ｕ／μＬ）
ＵＧＴ１Ａ１＊６　ｐｒｉｍｅｒ（Ｏｐｅｒｏｎ　Ｂｉｏｔｅｃｈｎｏｌｏｇｉｅｓ社製）
　Ｆｏｒｗａｒｄ（野生型）（１０ｐｍｏｌ／μＬ）：５’－（ｃｇｃｃｔｃｇｔｔｇｔａｃａｔｃａｇａｇｃｇｇ）－３’（配列番号１）
　Ｆｏｒｗａｒｄ（変異型）（１０ｐｍｏｌ／μＬ）：５’－（ｃｔｇａｃｇｃｃｔｃｇｔｔｇｔａｃａｔｃａｇａｇｃｇａ）－３”（配列番号２）
　Ｒｅｖｅｒｓｅ（１０ｐｍｏｌ／μＬ）：５’－（ｃａｃａｔｃｃｔｃｃｃｔｔｔｇｇａａｔｇｇｃａ）－３”（配列番号３）
Ｎｕｃｌｅａｓｅ－ｆｒｅｅ　Ｗａｔｅｒ（ｎｏｔ　ＤＥＰＣ－ｔｒｅａｔｅｄ）（Ａｍｂｉｏｎ社製、Ｌｏｔ．０８０３０１５）
ＵＧＴ１Ａ１遺伝子　野生型配列挿入プラスミド（１×１０６コピー／μＬ）
ＵＧＴ１Ａ１遺伝子　変異型配列挿入プラスミド（１×１０６コピー／μＬ） (AS-PCR amplification)
Wild-type and mutant amplification products were obtained under the following AS-PCR conditions.
(1) Reagent AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen, Lot. 760816)
10x AccuPrime PCR Buffer I
AccuPrime Taq DNA Polymerase High Fidelity (5 U / μL)
UGT1A1 * 6 primer (manufactured by Operon Biotechnologies)
Forward (wild type) (10 pmol / μL): 5 ′-(cgcctcgttgtatacatcaggcgg) -3 ′ (SEQ ID NO: 1)
Forward (mutant type) (10 pmol / μL): 5 ′-(ctgacccccctgtgtatacatgagcga) -3 ″ (SEQ ID NO: 2)
Reverse (10 pmol / μL): 5 ′-(cacatcctccccttttgagagtgga) -3 ″ (SEQ ID NO: 3)
Nuclease-free Water (not DEPC-treated) (Ambion, Lot.0803015)
UGT1A1 gene wild-type sequence insertion plasmid (1 × 10 6 copies / μL)
UGT1A1 gene mutant sequence insertion plasmid (1 × 10 6 copies / μL)
（２）調製
５μＬの１０×ＡｃｃｕＰｒｉｍｅ　ＰＣＲ　Ｂｕｆｆｅｒ　Ｉ、１μＬのＦｏｒｗａｒｄ　ｐｒｉｍｅｒ、１μＬのＲｅｖｅｒｓｅ　ｐｒｉｅｍｒに総量が４９μＬとなるようＮｕｃｌｅａｓｅ－ｆｒｅｅ　Ｗａｔｅｒで調整した溶液に、１μＬのＵＧＴ１Ａ１遺伝子配列挿入プラスミドを添加し反応溶液とした。 (2) Preparation 5 μL of 10 × AccuPrime PCR Buffer I, 1 μL Forward primer, 1 μL Reverse primer, and 1 μL UGT1A1 gene sequence added to the solution adjusted with Nuclease-free Water to a total volume of 49 μL It was set as the solution.
（３）反応
Ｃ１０００（バイオ・ラッド　ラボラトリーズ社製）を用いて、ＰＣＲ反応を行った。温度サイクルは以下に示す通りである。
９４℃、３０秒でテンプレートを熱変性させ、９４℃を１５秒、６２℃を１５秒、６８℃を３０秒の増幅サイクルを４０サイクル行い、最後に６８℃、５分で保温した。サンプルは使用するまで４℃で保存した。 (3) PCR reaction was performed using reaction C1000 (Bio-Rad Laboratories). The temperature cycle is as follows.
The template was thermally denatured at 94 ° C. for 30 seconds, 40 cycles of 94 ° C. for 15 seconds, 62 ° C. for 15 seconds, and 68 ° C. for 30 seconds were performed, and finally the temperature was kept at 68 ° C. for 5 minutes. Samples were stored at 4 ° C. until use.
ＡＳ－ＰＣＲ増幅後、電気泳動（アドバンス社製、「Ｍｕｐｉｄ－ｅｘ」）にて８０ｂｐ付近に増幅産物に由来するバンドを確認した。増幅産物の大きさは、２０ｂｐ　ＤＮＡ　Ｌａｄｄｅｒマーカー（タカラバイオ社製）を用いて確認した。 After AS-PCR amplification, a band derived from the amplified product was confirmed at around 80 bp by electrophoresis (manufactured by Advance, “Mupid-ex”). The size of the amplified product was confirmed using a 20 bp DNA Ladder marker (manufactured by Takara Bio Inc.).
（ＨＰＬＣ分析）
準備したアニオン交換カラムを用いて、以下の条件でＡＳ－ＰＣＲ増幅産物を分離検出した。
システム：ＬＣ－２０Ａシリーズ（島津製作所社製）
溶離液：溶離液Ａ　２５ｍｍｏｌ／Ｌトリス塩酸緩衝液（ｐＨ７．５）
　　　　溶離液Ｂ　２５ｍｍｏｌ／Ｌトリス塩酸緩衝液（ｐＨ７．５）＋１ｍｏｌ／Ｌグアニジン塩酸塩
分析時間：アニオン交換カラム１を用いたときの分析時間は１０分
　　　　　アニオン交換カラム２を用いたときの分析時間は２０分
溶出法：以下に示すグラジエント条件により、溶離液Ｂの混合比率を直線的に増加させた。
　　　　アニオン交換カラム１を用いたときの条件
　　　　０分（溶離液Ｂ４０％）→１０分（溶離液Ｂ５０％）
　　　　アニオン交換カラム２を用いたときの条件
　　　　０分（溶離液Ｂ７０％）→２０分（溶離液Ｂ９０％）
検体：ＵＧＴ１Ａ１＊６領域の野生型７６ｂｐ
　　　ＵＧＴ１Ａ１＊６領域の変異型７９ｂｐ　
流速：０．５ｍＬ／ｍｉｎ（アニオン交換カラム１を用いたとき）
　　　１．０ｍＬ／ｍｉｎ（アニオン交換カラム２を用いたとき）
検出波長：２６０ｎｍ
試料注入量：１０μＬ (HPLC analysis)
AS-PCR amplification products were separated and detected using the prepared anion exchange column under the following conditions.
System: LC-20A series (manufactured by Shimadzu Corporation)
Eluent: Eluent A 25 mmol / L Tris-HCl buffer (pH 7.5)
Eluent B 25 mmol / L Tris-HCl buffer (pH 7.5) +1 mol / L guanidine hydrochloride Analysis time: Analysis time when using anion exchange column 1 is 10 minutes Analysis time when using anion exchange column 2 is Elution method for 20 minutes: The mixing ratio of the eluent B was increased linearly under the gradient conditions shown below.
Conditions when using anion exchange column 1 0 min (eluent B 40%) → 10 min (eluent B 50%)
Conditions when using anion exchange column 2 0 min (eluent B 70%) → 20 min (eluent B 90%)
Specimen: Wild type 76 bp of UGT1A1 * 6 region
Mutation 79 bp of UGT1A1 * 6 region
Flow rate: 0.5 mL / min (when using anion exchange column 1)
1.0 mL / min (when using anion exchange column 2)
Detection wavelength: 260 nm
Sample injection volume: 10 μL
（参考例１）
参考例１では、ＵＧＴ１Ａ１＊６領域の野生型２７１ｂｐと変異型２７４ｂｐとの分離検出を行った。 (Reference Example 1)
In Reference Example 1, separation detection of wild type 271 bp and mutant type 274 bp in the UGT1A1 * 6 region was performed.
（ＡＳ－ＰＣＲ増幅）
以下に示すＡＳ－ＰＣＲ条件によって野生型と変異型の増幅産物を得た。 (AS-PCR amplification)
Wild-type and mutant amplification products were obtained under the following AS-PCR conditions.
（１）試薬
ＡｃｃｕＰｒｉｍｅ　Ｔａｑ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　Ｈｉｇｈ　Ｆｉｄｅｌｉｔｙ（Ｉｎｖｉｔｏｒｇｅｎ社製、Ｌｏｔ．７６０８１６）
１０×ＡｃｃｕＰｒｉｍｅ　ＰＣＲ　Ｂｕｆｆｅｒ　Ｉ
ＡｃｃｕＰｒｉｍｅ　Ｔａｑ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　Ｈｉｇｈ　Ｆｉｄｅｌｉｔｙ（５Ｕ／μＬ）
ＵＧＴ１Ａ１＊６　ｐｒｉｍｅｒ（Ｏｐｅｒｏｎ　Ｂｉｏｔｅｃｈｎｏｌｏｇｉｅｓ社製）
　Ｆｏｒｗａｒｄ（野生型）（１０ｐｍｏｌ／μＬ）：５’－（ｃｇｃｃｔｃｇｔｔｇｔａｃａｔｃａｇａｇｃｇｇ）－３’（配列番号１）
　Ｆｏｒｗａｒｄ（変異型）（１０ｐｍｏｌ／μＬ）：５’－（ｃｔｇａｃｇｃｃｔｃｇｔｔｇｔａｃａｔｃａｇａｇｃｇａ）－３”（配列番号２）
　Ｒｅｖｅｒｓｅ（１０ｐｍｏｌ／μＬ）：５’－（ｇａａａｇｇｇｔｃｃｇｔｃａｇｃａｔｇａｃ）－３”（配列番号４）
Ｎｕｃｌｅａｓｅ－ｆｒｅｅ　Ｗａｔｅｒ（ｎｏｔ　ＤＥＰＣ－ｔｒｅａｔｅｄ）（Ａｍｂｉｏｎ社製、Ｌｏｔ．０８０３０１５）
ＵＧＴ１Ａ１遺伝子　野生型配列挿入プラスミド（１×１０６コピー／μＬ）
ＵＧＴ１Ａ１遺伝子　変異型配列挿入プラスミド（１×１０６コピー／μＬ） (1) Reagent AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen, Lot. 760816)
10x AccuPrime PCR Buffer I
AccuPrime Taq DNA Polymerase High Fidelity (5 U / μL)
UGT1A1 * 6 primer (manufactured by Operon Biotechnologies)
Forward (wild type) (10 pmol / μL): 5 ′-(cgcctcgttgtatacatcaggcgg) -3 ′ (SEQ ID NO: 1)
Forward (mutant type) (10 pmol / μL): 5 ′-(ctgacccccctgtgtatacatgagcga) -3 ″ (SEQ ID NO: 2)
Reverse (10 pmol / μL): 5 ′-(gaaagggtccgtcagcatgac) -3 ″ (SEQ ID NO: 4)
Nuclease-free Water (not DEPC-treated) (Ambion, Lot.0803015)
UGT1A1 gene wild-type sequence insertion plasmid (1 × 10 6 copies / μL)
UGT1A1 gene mutant sequence insertion plasmid (1 × 10 6 copies / μL)
（２）調製
５μＬの１０×ＡｃｃｕＰｒｉｍｅ　ＰＣＲ　Ｂｕｆｆｅｒ　Ｉ、１μＬのＦｏｒｗａｒｄ　ｐｒｉｍｅｒ、１μＬのＲｅｖｅｒｓｅ　ｐｒｉｅｍｒに総量が４９μＬとなるようＮｕｃｌｅａｓｅ－ｆｒｅｅ　Ｗａｔｅｒで調整した溶液に、１μＬのＵＧＴ１Ａ１遺伝子配列挿入プラスミドを添加し反応溶液とした。 (2) Preparation 5 μL of 10 × AccuPrime PCR Buffer I, 1 μL Forward primer, 1 μL Reverse primer, and 1 μL UGT1A1 gene sequence added to the solution adjusted with Nuclease-free Water to a total volume of 49 μL It was set as the solution.
（３）反応
Ｃ１０００（バイオ・ラッド　ラボラトリーズ社製）を用いて、ＰＣＲ反応を行った。温度サイクルは以下に示す通りである。
９４℃、３０秒でテンプレートを熱変性させ、９４℃を１５秒、６２℃を１５秒、６８℃を３０秒の増幅サイクルを４０サイクル行い、最後に６８℃、５分で保温した。サンプルは使用するまで４℃で保存した。 (3) PCR reaction was performed using reaction C1000 (Bio-Rad Laboratories). The temperature cycle is as follows.
The template was thermally denatured at 94 ° C. for 30 seconds, 40 cycles of 94 ° C. for 15 seconds, 62 ° C. for 15 seconds, and 68 ° C. for 30 seconds were performed, and finally the temperature was kept at 68 ° C. for 5 minutes. Samples were stored at 4 ° C. until use.
ＡＳ－ＰＣＲ増幅後、電気泳動（アドバンス社製、「Ｍｕｐｉｄ－ｅｘ」）にて２７０ｂｐ付近（２００ｂｐと３００ｂｐの間）に増幅産物に由来するバンドを確認した。増幅産物の大きさは、２０ｂｐ　ＤＮＡ　Ｌａｄｄｅｒマーカー（タカラバイオ社製）を用いて確認した。 After AS-PCR amplification, a band derived from the amplified product was confirmed around 270 bp (between 200 bp and 300 bp) by electrophoresis (manufactured by Advance, “Mupid-ex”). The size of the amplified product was confirmed using a 20 bp DNA Ladder marker (manufactured by Takara Bio Inc.).
（ＨＰＬＣ分析）
準備したアニオン交換カラムを用いて、以下の条件でＡＳ－ＰＣＲ増幅産物を分離検出した。
システム：ＬＣ－２０Ａシリーズ（島津製作所社製）
溶離液：溶離液Ａ　２５ｍｍｏｌ／Ｌトリス塩酸緩衝液（ｐＨ７．５）
　　　　溶離液Ｂ　２５ｍｍｏｌ／Ｌトリス塩酸緩衝液（ｐＨ７．５）＋１ｍｏｌ／Ｌグアニジン塩酸塩
分析時間：アニオン交換カラム１を用いたときの分析時間は１０分
　　　　　アニオン交換カラム２を用いたときの分析時間は２０分
溶出法：以下に示すグラジエント条件により、溶離液Ｂの混合比率を直線的に増加させた。
　　　　アニオン交換カラム１を用いたときの条件
　　　　０分（溶離液Ｂ６０％）→１０分（溶離液Ｂ８０％）
　　　　アニオン交換カラム２を用いたときの条件
　　　　０分（溶離液Ｂ８０％）→２０分（溶離液Ｂ１００％）
検体：ＵＧＴ１Ａ１＊６領域の野生型２７１ｂｐ
　　　ＵＧＴ１Ａ１＊６領域の変異型２７４ｂｐ　
流速：０．５ｍＬ／ｍｉｎ（アニオン交換カラム１を用いたとき）
　　　１．０ｍＬ／ｍｉｎ（アニオン交換カラム２を用いたとき）
検出波長：２６０ｎｍ
試料注入量：１０μＬ (HPLC analysis)
AS-PCR amplification products were separated and detected using the prepared anion exchange column under the following conditions.
System: LC-20A series (manufactured by Shimadzu Corporation)
Eluent: Eluent A 25 mmol / L Tris-HCl buffer (pH 7.5)
Eluent B 25 mmol / L Tris-HCl buffer (pH 7.5) +1 mol / L guanidine hydrochloride Analysis time: Analysis time when using anion exchange column 1 is 10 minutes Analysis time when using anion exchange column 2 is Elution method for 20 minutes: The mixing ratio of the eluent B was increased linearly under the gradient conditions shown below.
Conditions when using anion exchange column 1 0 min (eluent B 60%) → 10 min (eluent B 80%)
Conditions when using anion exchange column 2 0 min (eluent B 80%) → 20 min (eluent B 100%)
Specimen: Wild type 271 bp of UGT1A1 * 6 region
Mutant 274 bp of UGT1A1 * 6 region
Flow rate: 0.5 mL / min (when using anion exchange column 1)
1.0 mL / min (when using anion exchange column 2)
Detection wavelength: 260 nm
Sample injection volume: 10 μL
（比較例１）
比較例１では、ＵＧＴ１Ａ１＊６領域の野生型７６ｂｐと変異型９６ｂｐとの分離検出を試みた。 (Comparative Example 1)
In Comparative Example 1, an attempt was made to separate and detect wild type 76 bp and mutant type 96 bp of the UGT1A1 * 6 region.
（１）試薬
ＡｃｃｕＰｒｉｍｅ　Ｔａｑ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　Ｈｉｇｈ　Ｆｉｄｅｌｉｔｙ（Ｉｎｖｉｔｏｒｇｅｎ社製、Ｌｏｔ．７６０８１６）
１０×　ＡｃｃｕＰｒｉｍｅ　ＰＣＲ　Ｂｕｆｆｅｒ　Ｉ
ＡｃｃｕＰｒｉｍｅ　Ｔａｑ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　Ｈｉｇｈ　Ｆｉｄｅｌｉｔｙ（５Ｕ／μＬ）
ＵＧＴ１Ａ１＊６　ｐｒｉｍｅｒ（Ｏｐｅｒｏｎ　Ｂｉｏｔｅｃｈｎｏｌｏｇｉｅｓ社製）
　Ｆｏｒｗａｒｄ（野生型）（１０ｐｍｏｌ／μＬ）：５’－（ｃｇｃｃｔｃｇｔｔｇｔａｃａｔｃａｇａｇｃｇｇ）－３’（配列番号１）
　Ｆｏｒｗａｒｄ（変異型）（１０ｐｍｏｌ／μＬ）：５’－（ａｔａｇｔｔｇｔｃｃｔａｇｃａｃｃｔｇａｃｇｃｃｔｃｇｔｔｇｔａｃａｔｃａｇａｇｃｇａ）－３”（配列番号５）
　Ｒｅｖｅｒｓｅ（１０ｐｍｏｌ／μＬ）：５’－（ｃａｃａｔｃｃｔｃｃｃｔｔｔｇｇａａｔｇｇｃａ）－３”（配列番号３）
Ｎｕｃｌｅａｓｅ－ｆｒｅｅ　Ｗａｔｅｒ（ｎｏｔ　ＤＥＰＣ－ｔｒｅａｔｅｄ）（Ａｍｂｉｏｎ社製、Ｌｏｔ．０８０３０１５）
ＵＧＴ１Ａ１遺伝子　野生型配列挿入プラスミド（１×１０６コピー／μＬ）
ＵＧＴ１Ａ１遺伝子　変異型配列挿入プラスミド（１×１０６コピー／μＬ） (1) Reagent AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen, Lot. 760816)
10x AccuPrime PCR Buffer I
AccuPrime Taq DNA Polymerase High Fidelity (5 U / μL)
UGT1A1 * 6 primer (manufactured by Operon Biotechnologies)
Forward (wild type) (10 pmol / μL): 5 ′-(cgcctcgttgtatacatcaggcgg) -3 ′ (SEQ ID NO: 1)
Forward (mutant) (10 pmol / μL): 5 ′-(atagtttgtcctagcacctacacccccctgtttatacatcaggcga) -3 ″ (SEQ ID NO: 5)
Reverse (10 pmol / μL): 5 ′-(cacatcctccccttttgagagtgga) -3 ″ (SEQ ID NO: 3)
Nuclease-free Water (not DEPC-treated) (Ambion, Lot.0803015)
UGT1A1 gene wild-type sequence insertion plasmid (1 × 10 6 copies / μL)
UGT1A1 gene mutant sequence insertion plasmid (1 × 10 6 copies / μL)
（２）調製
５μＬの１０×ＡｃｃｕＰｒｉｍｅ　ＰＣＲ　Ｂｕｆｆｅｒ　Ｉ、１μＬのＦｏｒｗａｒｄ　ｐｒｉｍｅｒ、１μＬのＲｅｖｅｒｓｅ　ｐｒｉｅｍｒに総量が４９μＬとなるようＮｕｃｌｅａｓｅ－ｆｒｅｅ　Ｗａｔｅｒで調整した溶液に、１μＬのＵＧＴ１Ａ１遺伝子配列挿入プラスミドを添加し反応溶液とした。 (2) Preparation 5 μL of 10 × AccuPrime PCR Buffer I, 1 μL Forward primer, 1 μL Reverse primer, and 1 μL UGT1A1 gene sequence added to the solution adjusted with Nuclease-free Water to a total volume of 49 μL It was set as the solution.
（３）反応
Ｃ１０００（バイオ・ラッド　ラボラトリーズ社製）を用いて、ＰＣＲ反応を行った。温度サイクルは以下に示す通りである。
９４℃、３０秒でテンプレートを熱変性させ、９４℃を１５秒、６２℃を１５秒、６８℃を３０秒の増幅サイクルを４０サイクル行い、最後に６８℃、５分で保温した。サンプルは使用するまで４℃で保存した。 (3) PCR reaction was performed using reaction C1000 (Bio-Rad Laboratories). The temperature cycle is as follows.
The template was thermally denatured at 94 ° C. for 30 seconds, 40 cycles of 94 ° C. for 15 seconds, 62 ° C. for 15 seconds, and 68 ° C. for 30 seconds were performed, and finally the temperature was kept at 68 ° C. for 5 minutes. Samples were stored at 4 ° C. until use.
ＡＳ－ＰＣＲ増幅後、電気泳動（アドバンス社製、「Ｍｕｐｉｄ－ｅｘ」）にて増幅産物の確認を行ったところ、非特異増幅と思われる多数のバンドが確認された。これは、ＡＳ－ＰＣＲ増幅が正常に行えなかったことを意味する。そのため、ＨＰＬＣ分析は実施しなかった。 After amplification of AS-PCR, amplification products were confirmed by electrophoresis (manufactured by Advance, “Mupid-ex”). As a result, many bands that seemed to be non-specific amplification were confirmed. This means that AS-PCR amplification could not be performed normally. Therefore, HPLC analysis was not performed.
（参考例２）
参考例２では、ＵＧＴ１Ａ１＊６領域の野生型７６ｂｐと変異型７９ｂｐとの分離検出を行った。 (Reference Example 2)
In Reference Example 2, separation detection of wild type 76 bp and mutant type 79 bp in the UGT1A1 * 6 region was performed.
溶離液Ｂに添加する塩をグアニジン塩酸塩に代えて塩化ナトリウムとしたこと以外は、実施例１と同様にして、アニオン交換カラム２を用いてＨＰＬＣ分析を行った。 HPLC analysis was performed using anion exchange column 2 in the same manner as in Example 1 except that sodium chloride was used instead of guanidine hydrochloride as the salt added to eluent B.
実施例１において、ＵＧＴ１Ａ１＊６領域の野生型７６ｂｐと変異型７９ｂｐとを分離検出して得られたクロマトグラムを図１（アニオン交換カラム１を用いた場合）と図２（アニオン交換カラム２を用いた場合）に示す。図１、２の結果から、両カラムともにＡＳ－ＰＣＲで増幅したＵＧＴ１Ａ１＊６領域の野生型７６ｂｐと変異型７９ｂｐを良好に分離検出することができた。特に、アニオン交換カラム１を用いた場合は、短時間でほぼ完全に分離検出することができた。 In Example 1, chromatograms obtained by separating and detecting wild type 76 bp and mutant type 79 bp of UGT1A1 * 6 region are shown in FIG. 1 (when anion exchange column 1 is used) and FIG. When used). From the results shown in FIGS. 1 and 2, both columns were able to successfully separate and detect wild-type 76 bp and mutant-type 79 bp of UGT1A1 * 6 region amplified by AS-PCR. In particular, when the anion exchange column 1 was used, almost complete separation and detection could be achieved in a short time.
参考例１において、ＵＧＴ１Ａ１＊６領域の野生型２７１ｂｐと変異型２７４ｂｐとを分離検出して得られたクロマトグラムを図３（アニオン交換カラム１を用いた場合）と図４（アニオン交換カラム２を用いた場合）に示す。図３、４の結果から、実施例１とは対照的に、ＡＳ－ＰＣＲで増幅したＵＧＴ１Ａ１＊６領域の野生型２７１ｂｐと変異型２７４ｂｐとを分離することができなかった。これは、ＡＳ－ＰＣＲ増幅産物のサイズに対して、野生型と変異型の鎖長差が小さかったためであると考えられる。 In Reference Example 1, chromatograms obtained by separating and detecting wild type 271 bp and mutant type 274 bp in the UGT1A1 * 6 region are shown in FIG. 3 (when anion exchange column 1 is used) and FIG. When used). From the results of FIGS. 3 and 4, in contrast to Example 1, it was not possible to separate the wild type 271 bp and the mutant type 274 bp of the UGT1A1 * 6 region amplified by AS-PCR. This is considered to be because the difference in chain length between the wild type and the mutant type was small with respect to the size of the AS-PCR amplification product.
参考例２において、アニオン交換カラム２を用いてＵＧＴ１Ａ１＊６領域の野生型７６ｂｐと変異型７９ｂｐとを分離検出して得られたクロマトグラムを図５に示す。溶離液Ｂにグアニジン塩酸塩に代えて塩化ナトリウムを添加すると、野生型７６ｂｐと変異型７９ｂｐを分離することができなかった。 FIG. 5 shows a chromatogram obtained by separating and detecting wild type 76 bp and mutant type 79 bp in the UGT1A1 * 6 region using the anion exchange column 2 in Reference Example 2. When sodium chloride was added to eluent B instead of guanidine hydrochloride, wild type 76 bp and mutant type 79 bp could not be separated.
本発明によれば、迅速かつ簡便な一塩基多型の検出方法を提供することができる。 According to the present invention, a rapid and simple method for detecting a single nucleotide polymorphism can be provided.

Claims (4)

  1. ＡＳ－ＰＣＲ法によって増幅された野生型と変異型の産物を、イオン交換クロマトグラフィーを用いて分析することを特徴とする一塩基多型の検出方法。 A method for detecting a single nucleotide polymorphism, characterized in that wild-type and mutant products amplified by an AS-PCR method are analyzed using ion-exchange chromatography.
  2. ＡＳ－ＰＣＲ法によって増幅された産物の大きさが２００ｂｐ以下であり、かつ、野生型と変異型の鎖長差が１０ｂｐ以下であることを特徴とする、請求項１記載の一塩基多型の検出方法。 The single nucleotide polymorphism according to claim 1, wherein the size of the product amplified by the AS-PCR method is 200 bp or less, and the difference in chain length between the wild type and the mutant is 10 bp or less. Detection method.
  3. イオン交換クロマトグラフィーにおいて、下記式（１）で示されるグアニジンから誘導されるグアニジン塩を含有する溶離液を用いることを特徴とする、請求項１又は２記載の一塩基多型の検出方法。
    Figure JPOXMLDOC01-appb-C000001
         The method for detecting a single nucleotide polymorphism according to claim 1 or 2, wherein an eluent containing a guanidine salt derived from guanidine represented by the following formula (1) is used in ion exchange chromatography.
    Figure JPOXMLDOC01-appb-C000001
  4. グアニジン塩は、グアニジン塩酸塩又はグアニジン硫酸塩であることを特徴とする、請求項３記載の一塩基多型の検出方法。
    　
    　     The method for detecting a single nucleotide polymorphism according to claim 3, wherein the guanidine salt is guanidine hydrochloride or guanidine sulfate.

PCT/JP2012/050430 2011-01-12 2012-01-12 Method for detecting single nucleotide polymorphisms WO2012096329A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP12734188.1A EP2664917B1 (en) 2011-01-12 2012-01-12 Method for detecting single nucleotide polymorphisms
KR1020137020841A KR102047650B1 (en) 2011-01-12 2012-01-12 Method for detecting single nucleotide polymorphisms
CN201280005010.3A CN103348242B (en) 2011-01-12 2012-01-12 The detection method of single nucleotide polymorphism
US13/979,256 US20140147842A1 (en) 2011-01-12 2012-01-12 Method for detecting single nucleotide polymorphisms
JP2012552752A JP6061381B2 (en) 2011-01-12 2012-01-12 Single nucleotide polymorphism detection method
US14/669,735 US9447460B2 (en) 2011-01-12 2015-03-26 Method for detecting single nucleotide polymorphisms

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2011-004216 2011-01-12
JP2011004216 2011-01-12
JP2011-080369 2011-03-31
JP2011080369 2011-03-31

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US13/979,256 A-371-Of-International US20140147842A1 (en) 2011-01-12 2012-01-12 Method for detecting single nucleotide polymorphisms
US14/669,735 Continuation US9447460B2 (en) 2011-01-12 2015-03-26 Method for detecting single nucleotide polymorphisms

Publications (1)

Publication Number Publication Date
WO2012096329A1 true WO2012096329A1 (en) 2012-07-19

Family

ID=46507221

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2012/050430 WO2012096329A1 (en) 2011-01-12 2012-01-12 Method for detecting single nucleotide polymorphisms

Country Status (6)

Country Link
US (2) US20140147842A1 (en)
EP (1) EP2664917B1 (en)
JP (1) JP6061381B2 (en)
KR (1) KR102047650B1 (en)
CN (1) CN103348242B (en)
WO (1) WO2012096329A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2012133834A1 (en) * 2011-03-31 2014-07-28 積水メディカル株式会社 Sample nucleic acid for single nucleotide polymorphism detection, PCR primer for preparation of single nucleotide polymorphism detection sample, and method for preparing sample for single nucleotide polymorphism detection used for ion exchange chromatography analysis
WO2015129916A1 (en) * 2014-02-28 2015-09-03 国立研究開発法人国立がん研究センター Method for determining prognosis of renal cell carcinoma
WO2017170101A1 (en) * 2016-03-31 2017-10-05 積水メディカル株式会社 Method for detecting single-base substitution using ion exchange chromatography
WO2019022132A1 (en) * 2017-07-26 2019-01-31 積水メディカル株式会社 Method for detecting mutant gene
WO2019064483A1 (en) * 2017-09-29 2019-04-04 積水メディカル株式会社 Method for detecting single base substitution using ion exchange chromatography

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3673990A4 (en) * 2017-08-25 2021-05-05 Sekisui Medical Co., Ltd. Chromatography packing for separation and/or detection of methylated dna
CN114577936A (en) * 2022-03-03 2022-06-03 中科谱研（北京）科技有限公司 Separation and detection method of beta-nicotinamide mononucleotide in capsule

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001330969A (en) 2000-05-23 2001-11-30 Sekisui Chem Co Ltd Apparatus for removing photoresist
JP2009524412A (en) * 2005-12-30 2009-07-02 アンスティテュ・パストゥール Differential amplification of mutant nucleic acids by PCR in a mixture of nucleic acids.
JP2010504738A (en) * 2006-09-26 2010-02-18 ジーイー・ヘルスケア・バイオサイエンス・コーポレイション Nucleic acid purification method using anion exchange

Family Cites Families (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6098988A (en) 1983-11-01 1985-06-01 Chemo Sero Therapeut Res Inst Purification of lpf-ha
CA2063855C (en) 1991-04-03 1997-08-26 Will Bloch Precision and accuracy of anion-exchange separation of nucleic acids
US5438128A (en) 1992-02-07 1995-08-01 Millipore Corporation Method for rapid purifiction of nucleic acids using layered ion-exchange membranes
ATE302208T1 (en) 1994-02-11 2005-09-15 Qiagen Gmbh METHOD FOR SEPARATING DOUBLE STRAND/SINGLE STRAND NUCLEIC ACID STRUCTURES
SE9600590D0 (en) 1996-02-19 1996-02-19 Pharmacia Biotech Ab Methods for chromatographic separation of peptides and nucleic acid and new high-affinity ion exchange matrix
US6265168B1 (en) * 1998-10-06 2001-07-24 Transgenomic, Inc. Apparatus and method for separating and purifying polynucleotides
JP2004514874A (en) * 1999-10-12 2004-05-20 トランスジエノミツク・インコーポレーテツド Detection of nucleic acid heteroduplex molecules by anion-exchange chromatography
US7264925B2 (en) 2000-08-30 2007-09-04 Avi Biopharma, Inc. Method for analysis of oligonucleotide analogs
CN1451762A (en) 2002-04-12 2003-10-29 刘湘军 Determination of SNP by different length of PCR products
JP2004180637A (en) * 2002-12-06 2004-07-02 Fuji Photo Film Co Ltd Apparatus for separation and purification of nucleic acid
JP4228041B2 (en) 2003-07-08 2009-02-25 東洋紡績株式会社 Nucleotide polymorphism detection method
JP4491276B2 (en) 2004-05-17 2010-06-30 日本製粉株式会社 Method and kit for detecting the presence of a single nucleotide polymorphism in a target DNA sequence
JP2006075126A (en) 2004-09-13 2006-03-23 National Institute Of Advanced Industrial & Technology Method for detecting monobasic mutation and probe for detection
JP5101492B2 (en) 2005-04-26 2012-12-19 サンド・アクチエンゲゼルシヤフト Production of recombinant protein by self-proteolytic cleavage of fusion protein
CN1880480A (en) 2006-04-12 2006-12-20 重庆医科大学 PCR method for detection of allele specific primer and use in detection of KLOTHO gene polymorphism
GB2443505B (en) * 2006-09-26 2008-12-31 Ge Healthcare Bio Sciences Nucleic acid purification method
CN101323852B (en) 2007-06-11 2013-01-23 北京东胜创新生物科技有限公司 Reagent box and method for genome extraction on automatic nucleic acid extraction workstation
US20090053719A1 (en) * 2007-08-03 2009-02-26 The Chinese University Of Hong Kong Analysis of nucleic acids by digital pcr
CN101899437B (en) * 2010-04-15 2012-04-25 浙江大学 Functional molecular marker for resistance identification of blight in Cucumis melo L. and application thereof
CN101899511B (en) 2010-07-09 2012-08-08 华中农业大学 Method for detecting bovine leukocyte adhesion deficiency by applying allele specific polymerase chain reaction (AS-PCR)
WO2012096327A1 (en) 2011-01-12 2012-07-19 積水メディカル株式会社 Eluent for ion-exchange chromatography, and method of analyzing nucleic acid chains
CN103443274A (en) 2011-03-31 2013-12-11 积水医疗株式会社 Sample nucleic acid for single nucleotide polymorphism detection purposes, pcr primer for preparing sample for single nucleotide polymorphism detection purposes, and method for preparing sample for single nucleotide polymorphism detection purposes wh

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001330969A (en) 2000-05-23 2001-11-30 Sekisui Chem Co Ltd Apparatus for removing photoresist
JP2009524412A (en) * 2005-12-30 2009-07-02 アンスティテュ・パストゥール Differential amplification of mutant nucleic acids by PCR in a mixture of nucleic acids.
JP2010504738A (en) * 2006-09-26 2010-02-18 ジーイー・ヘルスケア・バイオサイエンス・コーポレイション Nucleic acid purification method using anion exchange

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GAUDET M. ET AL.: "Allele-specific PCR in SNP genotyping", METHODS MOL BIOL., vol. 578, 2009, pages 415 - 424, XP009177497 *
NATURE, vol. 324, 1986, pages 163 - 166
See also references of EP2664917A4
TSUCHIHASHI Z. ET AL.: "Progress in high throughput SNP genotyping methods", PHARMACOGENOMICS J., vol. 2, no. 2, 2002, pages 103 - 110, XP008010361 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2012133834A1 (en) * 2011-03-31 2014-07-28 積水メディカル株式会社 Sample nucleic acid for single nucleotide polymorphism detection, PCR primer for preparation of single nucleotide polymorphism detection sample, and method for preparing sample for single nucleotide polymorphism detection used for ion exchange chromatography analysis
WO2015129916A1 (en) * 2014-02-28 2015-09-03 国立研究開発法人国立がん研究センター Method for determining prognosis of renal cell carcinoma
CN106062215A (en) * 2014-02-28 2016-10-26 国立研究开发法人国立癌研究中心 Method for determining prognosis of renal cell carcinoma
US10190172B2 (en) 2014-02-28 2019-01-29 National Cancer Center Method for determining prognosis of renal cell carcinoma
CN111057752A (en) * 2014-02-28 2020-04-24 国立研究开发法人国立癌研究中心 Application of ion exchange chromatography and application of PCR (polymerase chain reaction) primer
CN106062215B (en) * 2014-02-28 2020-09-22 国立研究开发法人国立癌研究中心 Method for determining prognosis of renal cell carcinoma
WO2017170101A1 (en) * 2016-03-31 2017-10-05 積水メディカル株式会社 Method for detecting single-base substitution using ion exchange chromatography
WO2019022132A1 (en) * 2017-07-26 2019-01-31 積水メディカル株式会社 Method for detecting mutant gene
WO2019064483A1 (en) * 2017-09-29 2019-04-04 積水メディカル株式会社 Method for detecting single base substitution using ion exchange chromatography
CN109863245A (en) * 2017-09-29 2019-06-07 积水医疗株式会社 Detection method is replaced using the single base of ion-exchange chromatography
US10626452B2 (en) 2017-09-29 2020-04-21 Sekisui Medical Co., Ltd. Method for detecting single base substitution using ion-exchange chromatography

Also Published As

Publication number Publication date
KR102047650B1 (en) 2019-11-22
KR20140041421A (en) 2014-04-04
US9447460B2 (en) 2016-09-20
JPWO2012096329A1 (en) 2014-06-09
CN103348242B (en) 2016-08-17
EP2664917A1 (en) 2013-11-20
JP6061381B2 (en) 2017-01-18
US20150197795A1 (en) 2015-07-16
US20140147842A1 (en) 2014-05-29
EP2664917B1 (en) 2017-09-06
CN103348242A (en) 2013-10-09
EP2664917A4 (en) 2015-01-07

Similar Documents

Publication Publication Date Title
JP6061381B2 (en) Single nucleotide polymorphism detection method
JP5930443B2 (en) Ion exchange chromatography packing for nucleic acid strand separation detection and ion exchange chromatography column for nucleic acid strand separation detection
JP6222639B2 (en) Method for detecting methylated DNA
WO2015129916A1 (en) Method for determining prognosis of renal cell carcinoma
WO2012133834A1 (en) Sample nucleic acid for single nucleotide polymorphism detection purposes, pcr primer for preparing sample for single nucleotide polymorphism detection purposes, and method for preparing sample for single nucleotide polymorphism detection purposes which can be used in ion exchange chromatographic analysis
WO2019039532A1 (en) Method for assessing risk of hepatocellular carcinoma
JP6977933B2 (en) Prognosis determination method for renal cell carcinoma
JP6090985B2 (en) Nucleic acid strand separation method
WO2016024634A1 (en) Method for assessing chromosomal dysfunction effective in diagnosis of imprinting diseases
JP5812464B2 (en) Target nucleic acid separation and detection method, target nucleic acid separation and detection packing material for ion exchange chromatography, and target nucleic acid separation and detection column for ion exchange chromatography

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12734188

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2012552752

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2012734188

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2012734188

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 20137020841

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 13979256

Country of ref document: US